Isolation of hemophilus bacteria



May 20, 1969 c. CONTRONI ET 3,445,339

ISOLATION OF HEMOPHILUS BACTERIA Filed Feb. 6, 1968 INVENTORS 00/00 (00700 11/44 000 0000 70/ /05 0. 00/0/00 5y00 0y 0055- BY SemmesaMS'emmes ATTORNEYS United States Patent 3,445,339 ISOLATION OF HEMOPHILUS BACTERIA Guido Controni, 5327 85th Ave., New Carrolton, Md.

20784; Waheed N. Khan, 1703 East-West Highway,

Apt. 503, Silver Spring, Md. 20910; James R. Patrick,

Mullinix Mill Road, Mount Airy, Md. 21771; and

Sydney Ross, 2831 Chesterfield Place NW., Washington, D.C. 20008 Filed Feb. 6, 1968, Ser. No. 703,480 Int. Cl. C12k 1/04 U.S. Cl. 195-1035 7 Claims ABSTRACT OF THE DISCLOSURE A method of isolating and discerning the presence of Hemophilus species in cerebral spinal fluid and body cavity fluid specimens, which is equally adapted to isolation and examination of these bacteria: meningococci; staphylococci; pneumococci. One principle application of the method lies in the ready identification of pathogenic Hemophilus influenzae, which are virtually always capsulated and nearly all belong to type B.

DESCRIPTION OF THE PRIOR ART In the past, the most widely used medium in clinical laboratories for the isolation of Hemophilus influenzae is chocolate agar generally containing hemin and Supplement B. Disadvantages for the recovery of H. influenzae are that chocolate agar, being a very rich medium, supports the growth of other organisms which often overgrow the more fastidious H. influenzae; many of these organisms resemble H. influenzae due to opacity of the chocolate agar. As is known, the pathogenic H. influenzae colonies are generally recognized by an iridescence, rendered difficult to observe by the opaqueness of the medium chocolate agar. Since iridescence is perhaps the most characteristic property which helps difl erentiatc H. influenzae from H. paraz'nfluenzae, it is for this reason that a new process which would not only further enhance the growth of H. influenzae, but also permit a distinction between the perspective hemophilic organisms becomes important.

Additionally, Supplement B may be denatured by heat during preparation of the medium, said denaturation being diflicult to detect without strict quality control of the media. In the past, the Levinthal and Fildes media have been considered as effective for the growth of H. influenzae, both said media being transparent and suitable therefore for the study of iridescence of Hemophilus species, but lack of erythrocytes in Levinthal and Fildes media make them inadequate for such pathogens which are identified by their hemolysis. Because both such media must be freshly made and because they are difficult to prepare in a clinical laboratory, it is impractical to maintain a fresh supply thereof. Accordingly, the present method is adapted to isolating especially the selected bacteria for a more practical and faster diagnosis of the organism in meningitides and bacteremias.

ice

2 BRIEF DESCRIPTION OF THE DRAWINGS The drawings represent in schematic the process of isolation, hereinafter described and claimed:

FIGURE 1 is a flow sheet of the process;

FIGURE 2 represents in schematic the effect created thereby.

DESCRIPTION OF THE PREFERRED EMBODIMENTS In this invention, the following steps are takento accomplish the desired result of isolating, for example, the bacteria H. influenzae.

Step I A Petri dish is prepared with a sheep blood agar medium. The blood agar base media is prepared by introducing approximately 40 grams of a blood agar base into a liter of distilled water, heated until dissolved and sterilized at 118 to 120 C. under 15 lbs. pressure for 15 minutes. When the medium has cooled to 45 to 50 C., 5 to 10% sterile defibrinated sheep blood is added, and the same applied in 20 ml. quantities to a Petri dish for solidification.

Step II The second step involves innoculation and streaking of the blood agar medium with a cerebral spinal fluid or body cavity fluid specimen, the details of streaking being described hereinafter.

Step Ill Under the third step, a saponin-yeast or saponin-yeast antibiotic-bacitracin disc of low concentration is prepared from a sterile filter paper assay disc, the preferred solution being a 5% saponion solution to which may be added a 5% yeast extract preferably rich in DPN as a source of enzyme growth, although the process may be attained without DPN and/or Without yeast. The purpose of the saponin is to hemolyze erythrocytes, forming a clear zone of hemolysis around the disc when applied to the sheep blood agar medium, prepared as above. The initial application of the disc is to break cells and release hemin as an X factor and DPN as a V factor; the effect herein is not only to create a clear zone of hemolysis around the disc, but also by the action of saponin to release DPN from erythrocytes making it available to H. influenzae to enhance growth of colonies thereof.

Step IV Fourthly, there is an incubation of the inoculated plate resulting in growth and isolation of the pathogenic bacteria, incubation being at 37 C. for a period of 8 to 12 hours under the influence of carbon dioxide, preferably for a period of 18 to 24 hours.

With reference to FIG. 2 of the drawings, there is illustrated in fragment a portion of a Petri dish having the sheep blood agar medium within. For practical purposes, the medium is approximately 3 mm. deep. Although not shown, an inoculated zone of the sheep blood agar medium exists in an horizontal zone immediately above and below and through the treated zones and 130. This zone of inoculation would include the following: H. influenzae; Diplococcus pneumoniae and the Alpha Streptococci. This zone has been inoculated in accordance with the procedure set forth hereinafter.

The present blood agar plate shows not only a zone of transparency 120 wherein stromatolysis and/or hemolysis releases DPN from the erythrocytes, but also a zone of inhibition 130 for the D. pneumoniae. The particular disc 132 within is saturated with optochin. In this zone of inhibition 130, one is able to differentiate between the D. pneumoniae and Alpha streptococci; whereas in the zone 120, both zones being within the same Petri dish and within the same blood agar medium, the zone of transparency 120 is created by the hemolytic action of the saponin disc 122, permits the observation of the bacteria H. influenzae. Noteworthy is the fact that the zone 130 is opaque whereas the zone 120 is essentially transparent permitting thereby the unique distinctive observations claimed herein.

As claimed hereinafter, the zone of hemolysis 120 is created by the saponin emanating from the disc, whereas bacitracin thereof inhibits the growth of other foreign bacteria or other gram positive bacteria. This then creates an area 120 which is purely selective for observation of the H. influenzae. The characteristic iridescence of the H. influenzae bacteria is thus totally accurately observed in the transparent zone, and the other areas of the same medium may be used for totally unrelated observations and discoveries.

Experiment A successful experiment herein resulted in the followmg:

Sterile saponin solution was prepared by adding 3.0 grams of saponin to 100 cc. NaCl that is of a strength which is non-hemolyzing. The saponin was dissolved by mild agitation without formation of bubbles and 5.0 grams of yeast extract were added. When the yeast extract had dissolved completely, the solution was filtered through a 0.20 micron plain membrane Nalgene filter. Sterile bacitracin (2.0 units) paper discs, one-quarter inch in diameter, were saturated with the prepared saponin solution for our closed cavity fluid study. The saturated discs were then dried at 37 C. for 3 to 4 hours and stored at 4 C. prior to use. Chocolate, sheep blood and Fildes agar plates concurrently used in this study were made according to procedures described in Diagnostic Bacteriology by W. R. Bailey and E. G. Scott, 1966 ed., The C. V. Mosby Co., St. Louis, Mo.

Thirty clinical specimens were studied. The criteria to select a patient was an initial positive CSF smear demonstrating Gram-negative or Gram-positive organisms. The spinal fluid was then streaked on a sheep blood agar plate and a saponin-yeast extract disc placed over the inoculum. In addition to the blood plate, two chocolate plates were streaked, one for regular culture and the other for direct sensitivities. The time lapse between obtaining the spinal fluid and plating was no longer than one hour and the specimens were refrigerated while waiting to be plated. Blood cultures were incubated as they were obtained and subcultured every 12-18 hours. All the inoculated plates were incubated at 37 C. in a CO environment.

Seventy-five strains of Hemophilus species previously isolated from nasopharyngeal cultures and resistant to 2.0 units of bacitracin were tested with 3% saponin soaked discs to check any inhibitory eitect of saponin. Also, all the strains of H. influenzae isolated from blood and CSF were tested similarly to determine saponin and bacitracin susceptibility. Preliminary studies using the saponin-yeastextract solution with low concentration bacitracin discs for the recovery of H. influenzae from nasopharyngeal cultures have also been successful.

The streaking method was as follows: Two crops of either CSF or blood were placed into the media near the edge of a Petri plate and allowed to be absorbed. The

plates were streaked continuously by free horizontal strokes of the inoculating needle from edge to edge of the plate. The inoculating needle was jabbed into the medium /3, /2 and A of the way at one side of the plate until reaching the lower edge of the plate.

The results obtained with our technique on blood and CSF are shown in Table I. Thirty isolations of H. influenzae, type B, were made and compared with growth on chocolate agar medium.

TABLE I.-COMPARISON 0F EFFICACY IN ISOLATIONS OF H. INFLUENZAE ON BLOOD AGAR WITH SAPONIN ANTIBIOTIC DISC AND CHOCOLATE AGAR Recovery of H. influenzae type B on sheep blood agar using saponin antibiotic discs was 100%. Growth around the saponin antibiotic disc was heavy with numerous individual colonies or confluent where no distinction of colonies could be made. The individual colonies varied from 1 to 2 mm. in diameter and were raised, transparent and mucoid. The confluent growth was equally raised, mucoid and transparent but limited to the Zone of saponin stromatolysis (FIG. 2). The growth in both cases had the distinct mousy odor which is so characteristic of H. influenzae and although organism recovery was limited to the Zone of saponin stromatolysis, a quantitative cornparison of the number of colonies per unit area was comparable to a similar zone in a well prepared chocolate agar plate. By comparison, growth on chocolate agar under the same conditions was irregular and recovery was Three chocolate plates failed to grow H. influenzae and three had only scant growth.

Observation of iridescence was made on all the isolations made on blood agar with the impregnated discs. All the strains isolated were smooth and thereby classified as typical. Iridescence is perhaps the most characteristic property which helps differentiate H. influenzae from H. parainfluenzae. (Variation and Type Specificity in Bacterial Species Hemophilus influenzae. Pittman, M. J. Exp. Med., 53:471-492, 1931.) This observation of iridescence could not be made with chocolate plates.

Saponin discs did not have an inhibitory effect on the Hemophilus strains isolated from the nasopharyngeal cultures.

Whereas this process has been described in terms of H. influenzae isolation, it is practically adaptable to isolation and identity of numerous other bacteria. Accordingly and without especial modification, the invention is limited solely as claimed.

l-lemolyzing agents, not inhibitory to Hemophilus herein shall be construed to include detergents such as:

(A) Saponins 1 of the natural plant source; and

(B) Chemical detergents such as, for example only:

( 1) Saponin 1 (2) Micro (Made by International Products Corp, PO. Box 118, Trenton l, N.J.), active ingredient of this product is sodium ethylenediaminetetraacetate (Na C1 H1 g N2 (3) Sklar Kleen (J. Sklar Mfg. Co., Inc., Long Island City, N.Y.)

(4) Haemo-Sol (Aloe Medical Co., St. Louis, Mo.)

(5) Tweens, trademark for a series of surface active agents consisting of partial esters of longchain fatty acids with polyoxyalkylene derivatives of hexitol anhydride We claim:

1. A method of isolating microbes dependent upon X and/ or V factors of the genus Hemophilus while provid- 1 Saponiu contains a sapogeuon which may be a steroid or a tri-trpeue and a sugar moity: glucose, galactosc, peutosc or a methyl pentose.

ing also a substrate which differentiates bacteria by their characteristic morphology and hemolytic activity on such substrate, comprising the steps of:

(A) establishing a blood agar substrate from difibrinated blood;

(B) streaking said substrate with a suspected specimen;

(C) establishing a carrier which contains bacitracin and an hemolyzing agent which is non-inhibitory to Hemophili;

(D) placing said carrier upon the streaked substrate to promote growth of such bacteria in the zone of stromatolysis;

(E) incubating the streaked substrate with the carrier thereon.

2. The method of claim 1 in which the hemolyzing agent is a saponin.

3. The method of claim 1 in which the blood of the agar is sheep blood and in which the carrier is saturated with a saponin-yeast extract.

4. The method according to claim 3 wherein the yeast extract is rich in DPN.

References Cited B.B.L. Products, Culture Media, Materials and Apparatus for the Microbiological Laboratory, Baltimore Biological Laboratory Inc., 4th ed., 1956.

Oxoid Culture Media and Laboratory Preparations, Consolidated Laboratories Inc., Chicago Heights, 111., 1961.

ALVIN E. TANENHOLTZ, Primary Examiner.

US. Cl. X.R. 195-100 

